U.S. patent number 11,286,447 [Application Number 17/014,786] was granted by the patent office on 2022-03-29 for compositions and methods for disaggregation of biological tissue samples.
This patent grant is currently assigned to CLAREMONT BIOSOLUTIONS LLC. The grantee listed for this patent is CLAREMONT BIOSOLUTIONS LLC. Invention is credited to Gary Fife Blackburn, Mark Brown, Robert Doebler.
United States Patent |
11,286,447 |
Brown , et al. |
March 29, 2022 |
Compositions and methods for disaggregation of biological tissue
samples
Abstract
Devices and methods for the efficient disaggregation of tissue
samples, separating the tissue into individual intact cells or
small aggregates of cells for analysis. A device may include a
chamber to receive fluid and a tissue specimen containing more than
one cell to be disaggregated. The chamber may include an opening
and an agitator in fluid contact with the fluid and the tissue
specimen. The agitator may include a micromotor which provides
rotational motion to a shaft and an impeller fixed to the shaft
such that the impeller and the shaft rotate together upon provision
of the rotational motion by the micromotor. The device may include
an electrical energy source electrically coupled to the micromotor
to rotate the impeller sufficient to disaggregate the one or more
individual cells from the tissue specimen and in a manner which
does not lyse the one or more individual cells.
Inventors: |
Brown; Mark (Pasadena, CA),
Doebler; Robert (Upland, CA), Blackburn; Gary Fife
(Glendora, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
CLAREMONT BIOSOLUTIONS LLC |
Upland |
CA |
US |
|
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Assignee: |
CLAREMONT BIOSOLUTIONS LLC
(Upland, CA)
|
Family
ID: |
57126252 |
Appl.
No.: |
17/014,786 |
Filed: |
September 8, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210230523 A1 |
Jul 29, 2021 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15566158 |
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10801001 |
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PCT/US2016/027291 |
Apr 13, 2016 |
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62146876 |
Apr 13, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M
3/08 (20130101); C12M 45/02 (20130101); F05D
2270/03 (20130101); F05D 2250/82 (20130101) |
Current International
Class: |
G01N
1/00 (20060101); C12M 3/08 (20060101); C12M
1/33 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
International Search Report and Written Opinion, dated Jul. 26,
2016, for International Application No. PCT/US16/27291, 8 pages.
cited by applicant.
|
Primary Examiner: Nagpaul; Jyoti
Attorney, Agent or Firm: Seed Intellectual Property Law
Group LLP
Claims
The invention claimed is:
1. An apparatus to simultaneously or serially treat a plurality of
multi-cellular tissue specimens and to release one or more intact
individual cells in one or more of the plurality of multi-cellular
tissue specimens to permit subsequent analysis thereof, the
apparatus comprising: a plurality of spaced-apart chambers, each
chamber of the plurality of chambers configured to receive fluid
and a tissue specimen containing more than one cell to be
disaggregated to release one or more individual cells separate from
the tissue specimen, and each chamber comprising at least a first
opening to provide fluid communication with the chamber and to
receive the tissue specimen; a multi-agitator comprising a
plurality of spaced-apart agitators, each of the plurality of
agitators comprising: a micromotor which provides rotational motion
to a shaft extending from an interior of the micromotor, and an
impeller fixed to the shaft such that the impeller and the shaft
rotate together upon provision of the rotational motion by the
micromotor; and an electrical energy source electrically coupled to
the micromotors of each of the plurality of agitators, wherein, for
each of the micromotors, the electrical energy source provides
electrical energy to the micromotor sufficient to rotate the shaft
and the impeller in a manner sufficient to disaggregate the one or
more individual cells from the tissue specimen and in a manner
which does not lyse the one or more individual cells, wherein the
plurality of agitators are reversibly and simultaneously receivable
within respective ones of the first openings in the plurality of
chambers such that, for each agitator, the shaft and the impeller
contact the fluid and the tissue specimen in the respective
chamber, and wherein the micromotors are at least partially
received in the respective first openings in the chambers to seal
the first openings in use and are accessible to the respective
fluid and tissue specimen when the electrical energy is provided by
the electrical energy source.
2. The apparatus of claim 1 wherein the plurality of agitators of
the multi-agitator are configured in a linear array.
3. The apparatus of claim 1 wherein the plurality of agitators of
the multi-agitator are configured in a linear array of eight
agitators or a linear array of twelve agitators.
4. The apparatus of claim 1 wherein the plurality of chambers and
the plurality of agitators of the multi-agitator are each
configured in a linear array having 9 millimeter spacing.
5. The apparatus of claim 1 wherein the plurality of spaced-apart
chambers comprises a 96-well plate.
6. The apparatus of claim 1 wherein the plurality of spaced-apart
chambers comprises a linear array of spaced-apart chambers or a
two-dimensional array of spaced-apart chambers.
7. The apparatus of claim 1 wherein, for each of the micromotors,
the electrical energy source provides electrical energy to the
micromotor sufficient to disaggregate the one or more individual
cells from the tissue specimen in a manner which does not lyse more
than 5% of the cells in the multi-cellular tissue specimen.
8. The apparatus of claim 1 wherein, for each of the micromotors,
the electrical energy source provides electrical energy to the
micromotor sufficient to disaggregate the one or more individual
cells from the tissue specimen in a manner which does not lyse more
than 10% of the cells in the multi-cellular tissue specimen.
9. The apparatus of claim 1 wherein, for each of the micromotors,
the electrical energy source provides electrical energy to the
micromotor sufficient to disaggregate the one or more individual
cells from the tissue specimen in a manner which does not lyse more
than 20% of the cells in the multi-cellular tissue specimen.
10. The apparatus of claim 1 wherein the electrical energy source
provides a DC voltage of less than 2.0 volts to each of the
micromotors.
11. The system of claim 1 wherein the electrical energy source
provides a voltage waveform to each of the micromotors selected
from the group consisting of: a sine wave, a square wave, a
triangle wave, and a combination of a sine wave, square wave, and a
triangle wave.
12. A method, comprising: for each of a plurality of spaced-apart
chambers, placing a multi-cellular tissue specimen and a fluid in
the chamber via a first opening in each of the chambers;
positioning a multi-agitator in fluid contact with the fluid and
the tissue specimen in each of the plurality of spaced-apart
chambers, the multi-agitator comprising a plurality of spaced-apart
agitators, each of the plurality of agitators comprising: a
micromotor which provides rotational motion to a shaft extending
from an interior of the micromotor, and an impeller fixed to the
shaft such that the impeller and the shaft rotate together upon
provision of the rotational motion by the micromotor; and applying
electrical energy to the micromotors of the multi-agitator with an
electrical energy source electrically coupled to each of the
micromotors, for each micromotor, the electrical energy sufficient
to rotate the shaft and the impeller in a manner sufficient to
disaggregate one or more individual cells from the tissue specimen
and in a manner which does not lyse the one or more individual
cells, wherein at least a portion of each of the micromotors is
removably received in the respective first openings of the chambers
and forms a sealing arrangement to seal the first openings in
use.
13. The method of claim 12 wherein applying electrical energy to
the micromotors of the multi-agitator comprises applying electrical
energy to each of the micromotors serially.
14. The method of claim 12 wherein applying electrical energy to
the micromotors of the multi-agitator comprises applying electrical
energy to each of the micromotors simultaneously.
15. The method of claim 12 wherein applying the electrical energy
comprises applying electrical energy sufficient to rotate the shaft
and the impeller of each of the micromotors in a manner sufficient
to disaggregate the one or more individual cells from the tissue
specimen and in a manner which does not lyse more than 5% of the
cells in the multi-cellular tissue specimen in each of the
chambers.
16. The method of claim 12 wherein applying the electrical energy
comprises applying electrical energy sufficient to rotate the shaft
and the impeller of each of the micromotors in a manner sufficient
to disaggregate the one or more individual cells from the tissue
specimen and in a manner which does not lyse more than 10% of the
cells in the multi-cellular tissue specimen in each of the
chambers.
17. The method of claim 12 wherein applying the electrical energy
comprises applying electrical energy sufficient to rotate the shaft
and the impeller of each of the micromotors in a manner sufficient
to disaggregate the one or more individual cells from the tissue
specimen and in a manner which does not lyse more than 20% of the
cells in the multi-cellular tissue specimen in each of the
chambers.
18. The method of claim 12, further comprising performing molecular
combing analysis on at least one of the disaggregated one or more
individual cells.
19. The method of claim 12, further comprising performing nucleic
acid sequencing of at least one of the disaggregated one or more
individual cells.
20. The method of claim 12 wherein applying the electrical energy
comprises applying a voltage waveform selected from the group
consisting of: a sine wave, a square wave, a triangle wave, and a
combination of a sine wave, square wave, and a triangle wave.
Description
BACKGROUND
Technical Field
The present disclosure relates to preparation of samples for
single-cell analysis. Analysis of multi-celled samples, e.g.,
tissue samples, requires that the cells be separated from one
another if single cell analysis is desired. The present disclosure
describes devices which aid in such tissue disaggregation or tissue
dispersion. The present disclosure also relates to methods and
devices for the lysis of cells and extraction of DNA in a cell for
suitable for long-read sequencing and molecular combing
analysis.
Description of the Related Art
The biology of cells is typically examined in cell monolayer
culture applications, however, they have inherent limitations for
studying the effects of and screening for drugs and predicting in
vivo physiological responses (Girard et al., 2013, PLoS ONE,
8(10):e75345). As is known in the art, in vitro single cells or
cell monolayer behave very differently from an in vivo organization
of cells, wherein the cells are organized in a sophisticated
cellular network and form tissues. In those networks, cellular
responses of individual cells to drugs may be, at least to a
certain extent, controlled by its extracellular environment within
such network or tissue. Such extra-cellular environment, for
example, includes cell-cell interaction and cell-matrix
interactions. Particularly, cell-matrix interactions play an
important role in the formation of tumors and progression of
tumors.
Tumor cell aggregates are believed to exhibit specific
characteristic traits of their in vivo tumor counterparts. Through
their more realistic demonstration of a tumor's in vivo
architecture, cell-cell interactions and cell-matrix interactions,
they provide more valuable information regarding the cellular
differentiation, proliferation, apoptosis and gene expression of
the tumor cells in question (Kim et al., 2004, Breast Cancer
Research and Treatment, 85:281-291). Additionally, the use of tumor
cell aggregates or tumor spheroids in drug screening assays allows
one to observe the important interactions and behaviors of
different cell types, and in particular, stroma cells.
For the reasons discussed above, it is particularly desirable to
provide for drug validation and drug screening assays using cell
aggregates or tissue fragments, which mimic more the physiological
environment from where they are obtained than single cells. As
such, there is a long felt need in the art to provide compositions
and methods for the preparation of cell aggregates and/or tissue
fragments which more accurately reflect the in vivo structure of a
tissue, and more specifically, the in vivo structure of a cancerous
tissue. The present disclosure provides compositions and methods
useful for the processing of tissues and for the generation of a
plurality of cells, a plurality of cell aggregates and/or tissue
fragments. Tissues processed according to the present disclosure
can be used in various assay systems, including, but not limited
to, drug validation assays, drug screening assays, proliferation
assays, metabolic assays, metastasis assays, angiogenesis assays,
binding assays, biochemical assays, cellular assays, genetic
assays, and the like.
Cancer is the second leading cause of death in the Western World,
but is rapidly rising worldwide and is expected to become the
number one killer in a few years. Thus, there is tremendous need to
improve our understanding and ability to treat this deadly disease.
Nearly all cancer types form solid tumors, abnormal tissue masses
that are highly complex and dynamic. Recent evidence has pointed to
a model in which tumors can be viewed as an ecosystem including a
diverse array of cell types that work in concert to maintain
homeostasis and drive further development. This intra-tumor
cellular heterogeneity has been identified as a key factor
underlying progression, metastasis, and the development of drug
resistance. Cell types can include neoplastic subpopulations with
distinct genotypes and phenotypes that are generated through clonal
evolution, differentiation from rare stem-like precursors/cancer
stem cells, or most likely a combination of the two mechanisms.
Host cells of diverse origins, including non-tumor epithelium,
stroma, and immune subtypes, can also assist the tumor in different
capacities. Thus, analyzing tumor heterogeneity and identifying the
presence of key cell types have become major focus areas in tumor
biology and clinical diagnostics. Knowledge of different cell types
can also drive patient-specific protocols for cancer treatment.
A major challenge for solid tumor analysis is the fact that
specimens are three-dimensional tissue structures. This is
particularly true to assessing cellular heterogeneity and
identifying rare cell types such as cancer stem cells. Tissue-based
analysis methods such as histology, immunohistochemistry, and
fluorescence in-situ hybridization are clinical standards that
provide morphological and sub-cellular detail, but are low
throughput and detection signals are difficult to quantitate and
multiplex. Techniques that involve sample destruction such as
genome/transcriptome sequencing, microarrays, mass spectrometry,
and Western blotting can provide large amounts of molecular
information but retain no context with respect to the cellular
components in the original sample. Due to these limitations,
researchers and clinicians are increasingly employing cell-based
analysis platforms such as flow cytometry because they offer
high-throughput and multiplexed information about each cell within
the sample. Cell sorting can also be used to isolate rare cell
types such as cancer stem cells, metastatic precursors, and drug
resistance clones for additional study. The disadvantage is that
the tissue must first be broken down into single cells, which
requires considerable expenditure of time and effort. Moreover,
dissociation can potentially damage or otherwise bias samples.
Thus, tissue dissociation remains a major barrier to the
application of single cell techniques to solid tumor specimens.
Tumor tissue is currently dissociated into single cells using
proteolytic enzymes that digest cellular adhesion molecules and/or
the underlying extracellular matrix. The tumor tissue specimen is
first minced with a scalpel into approximately 1-2 mm pieces. The
enzyme or enzyme cocktail of choice is then applied. Trypsin is a
broadly reactive protease that is highly efficient, requiring only
short incubation times on the order of 15 minutes. Unfortunately,
trypsin can also cleave cell surface proteins that may provide
important diagnostic information or regulate cell function. For
example, it has been shown that CD44, a commonly used cancer stem
cell marker, is cleaved by trypsin resulting in significantly
reduced expression. Collagenase is a milder alternative that
digests collagen within the underlying extracellular matrix,
leaving cells largely undisturbed. For this reason, collagenase has
been employed for identifying and isolating cancer stem cells via
CD44 or other biomarkers. However, collagenase requires long
incubation times on the order of 1 to 2 hours that could negatively
affect cell viability or molecular expression. Non-enzymatic
options such as the calcium chelator ethylenediaminetetraacetic
acid (EDTA) can also be employed, but EDTA is much less efficient
and therefore used only to augment protease digestion. Following
initial enzymatic or chemical treatment procedure, samples are
subjected to fluid shear forces to mechanically liberate individual
cells. This is typically achieved by vortexing and/or repeatedly
pipetting the sample. These methods generate poorly defined shear
flow environments that do not allow control over sample exposure,
potentially resulting in variations across different batches or
laboratories. The gentleMACS.TM. Dissociator (Miltenyl Biotec) is a
commercial system that has been developed to standardize mechanical
dissociation, but its use with tumor specimens is not common and
performance is not well documented.
A final step that is used in many dissociation processes is to
remove large aggregates that remain by filtering, which results in
loss of sample. Taken together, tumor tissue dissociation involves
multiple manual processing steps that are time-consuming and labor
intensive, and there are numerous areas for which the resulting
cell suspension can be improved. Notably, enzymatic digestion is
either harsh or very long, large aggregates are lost to filtering,
and there is no way to control whether the recovered sample
contains single cells versus small clusters. Thus, new technology
and methodology development is critically needed to meet all of the
following goals: (1) improve dissociation efficiency so that the
entire sample is recovered as single cells, (2) maximize overall
cell quality in terms of viability and molecular biomarker
expression, (3) decrease processing time from hours to minutes, and
(4) automate the entire workflow to enable point-of-care operation
and direct connection to additional downstream tasks.
BRIEF SUMMARY
There is a need for methods and devices to provide rapid and simple
disaggregation of tissue and/or cell aggregates to produce
suspensions of individual cells to allow for analysis of cells
independently.
For example, cancer tissue is generally a heterogeneous population
of several types of cells, often at different stages of
progression. There is a need for better methods and devices to
allow such cancer tissues to be separated from one another for
analysis. The individual cells may be examined visually using a
microscope. Often such cells are stained by any of many techniques
known in the literature in order to determine particular
characteristics of the cells. Often, the cells are classified and
counted as a means of characterizing the cancer tissue. Such could
not be readily performed unless the tissue was disaggregated.
Another method for characterizing a cancer tissue by characterizing
the individual cells of the tissue involves performing nucleic acid
sequencing of individual cells from the population. Such methods
allow the heterogeneity of the tissue to be better understood and
can be used to target particular treatment strategies dependent
upon the characteristics of the tissue.
Individual cells can also be characterized by performing
reverse-transcriptase PCR of mRNA in the cell and determining the
gene expression level for genes or sequences of interest. Often,
nucleic acid microarray techniques are employed to measure the
relative concentration of many different mRNAs. Compilation of such
information for many individual cells of a particular cancer tissue
provides invaluable information to aid, for example, physicians to
apply more optimal treatment to the patients.
Tissue of various types can also be characterized by disaggregating
the tissue and then analyzing individual cells by flow cytometry.
In such techniques, labeled nucleic acid probes, labeled
antibodies, and stains are often used, sometimes simultaneously, to
allow more specific characterization of each cell type and the
population density of each cell class.
Techniques such as those described, as well as many others known to
those skilled in the art, all benefit from the ability to
effectively separate the cells of a tissue into individual cells.
Beneficially, the methods for disaggregation should be relatively
indiscriminate as to cell type so that the population of released
cells is representative of the population of the original tissue.
Methods and devices to disaggregate tissue should also do so with
as little damage or change to the morphology of the cells so that
their properties are as similar as practical to their state in the
tissue. It is also important form most analysis techniques that the
cell membranes of the cells be kept intact, i.e., the cells are not
lysed to any great extent, to allow more accurate analysis of the
biomolecules inside the cell membrane. Further, when the tissue is
fresh and the tissue cells are living, it is often important that
the disaggregation methods do not kill the cells to any appreciable
extent.
Described herein are methods and devices which provide rapid and
simple disaggregation of tissue sample or of multicellular
specimens of biological material.
In general, the methods described herein use novel devices which
incorporate micromotors to provide mechanical energy to fluids
containing the tissue specimens. Such micromotors are commonly
employed in cell phones to provide vibration of the phone. Such
micromotors are also commonly employed in toys and robotic devices,
for example, in model helicopters and model boats. The micromotors
are generally operated by application of a dc voltage to the
terminals of the micromotor. Most applications of the micromotors
are for battery powered devices and the micromotors are, therefore,
generally designed to operate at a voltage of 1.5 volts or greater.
The micromotors are generally cylindrical in shape and usually have
a diameter of less than 15 millimeters (mm). Because the
micromotors are small, they generally co not deliver very much
torque, but do rotate at very high rate. Rotation of up to 50,000
revolutions per minute (rpm) or greater are possible. The rotation
speed of the micromotors is generally dependent on the voltage
applied. Most applications, however, use the micromotors at the
upper end of their speed rating and, thus, at the upper end of
their recommended voltage specification. Because the motors are
designed and manufactured to operate at high rotational speeds,
they typically provide only low torque. As a consequence, the
motors may not begin to turn if the shaft extending from the
cylindrical body is in contact with another object. Similarly, the
motors stop turning easily if the shaft makes contact with another
object.
Because of the low torque nature of micromotors, there can be no
significant seal mechanism around the shaft to keep fluids from
entering the body of the motor. All attempts to encircle the shaft
with a fluid barrier such as a grommet or O-ring have resulted in
significantly reduced speeds, or more commonly, inability to turn
on the motor to rotate.
In some implementations of the present disclosure, the motors are
used in direct contact with the fluid and/or tissue specimen,
hence, without any fluid barrier to impede the rotation of the
shaft and impeller. This configuration allows the motor to operate
at high RPMs to drive the shaft and impeller at high speed.
Surprisingly, the motors continue to operate in direct contact with
the fluid.
In one or more implementations of the present disclosure, the shaft
and metal of the motor are treated with a silane compound such as
HMDS (hexamethyldisilizane) or alkylsilane. As is understood by
those skilled in the art, such treatment causes a reaction between
the silane and the metal surface to form a very thin layer on the
surface of the metal. In general, it was found that treatment with
hydrophobic silanes such as HMDS or alkylsilane impedes the
penetration of fluid into the motor body and thus improves the
performance of the motor in direct contact with the fluid. It is
assumed that the silane treatment forms a hydrophobic barrier and
that the high surface tension of water does not allow it to pass
the narrow passage between the shaft and the motor body.
Disaggregation of multicellular specimens is often also referred to
as dispersion or separation. These terms are used interchangeably
in this description.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
In the drawings, identical reference numbers identify similar
elements or acts. The sizes and relative positions of elements in
the drawings are not necessarily drawn to scale. For example, the
shapes of various elements and angles are not necessarily drawn to
scale, and some of these elements may be arbitrarily enlarged and
positioned to improve drawing legibility. Further, the particular
shapes of the elements as drawn, are not necessarily intended to
convey any information regarding the actual shape of the particular
elements, and may have been solely selected for ease of recognition
in the drawings.
FIG. 1 is a schematic view of a disaggregation apparatus comprising
an agitator and a container, according to one illustrated
implementation.
FIG. 2 is a schematic view of a disaggregation apparatus comprising
an integrated container and agitator, according to one illustrated
implementation.
FIGS. 3a-3b are sectional views of an impeller used in one or more
implementations of the present disclosure, according to one
illustrated implementation.
FIG. 4 shows photomicrographs of a disaggregated suspension of
chick embryo tissue, according to one illustrated
implementation.
FIG. 5 shows photomicrographs of a disaggregated suspension of
chick heart tissue, according to one illustrated
implementation.
DETAILED DESCRIPTION
In the following description, certain specific details are set
forth in order to provide a thorough understanding of various
disclosed implementations. However, one skilled in the relevant art
will recognize that implementations may be practiced without one or
more of these specific details, or with other methods, components,
materials, etc. In other instances, well-known structures
associated with computer systems, server computers, and/or
communications networks have not been shown or described in detail
to avoid unnecessarily obscuring descriptions of the
implementations. In other instances, methods commonly known for use
with and manipulation of tissue, cells, nucleic acids, proteins,
polypeptides, and other biological materials have not been
described, as they would be readily available to those of ordinary
skill in the art of such materials.
Unless the context requires otherwise, throughout the specification
and claims that follow, the word "comprising" is synonymous with
"including," and is inclusive or open-ended (i.e., does not exclude
additional, unrecited elements or method acts).
Reference throughout this specification to "one implementation" or
"an implementation" means that a particular feature, structure or
characteristic described in connection with the implementation is
included in at least one implementation. Thus, the appearances of
the phrases "in one implementation" or "in an implementation" in
various places throughout this specification are not necessarily
all referring to the same implementation. Furthermore, the
particular features, structures, or characteristics may be combined
in any suitable manner in one or more implementations.
As used in this specification and the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. It should also be noted that
the term "or" is generally employed in its sense including "and/or"
unless the context clearly dictates otherwise.
The headings and Abstract of the Disclosure provided herein are for
convenience only and do not interpret the scope or meaning of the
implementations.
A number of implementations of disaggregation apparatus, systems
and methods of use are described herein. The disaggregation
apparatus and systems perform disaggregation on a tissue using
mechanical agitation, to produce single cells. The material to be
disaggregated may take the form of solid biological materials, for
example cancer biopsies, cancer tissue, normal tissue, blood,
cervical swab samples, plant tissue, formalin-fixed,
paraffin-embedded (FFPE) samples, fresh frozen samples, fine needle
aspirate samples, etc.
In one general implementation, the disaggregation apparatus
includes a container such as a micro-centrifuge tube and an
agitation device. The agitation device is sized so that a portion
of it can be inserted into the opening of the container and
comprises a micromotor having a shaft at one end and an impeller
fitted on the end of the shaft. The micromotor can be connected to
a voltage or current source to drive the micromotor.
To perform disaggregation, the tissue to be disaggregated is placed
in the container, generally with a fluid such as saline that is
compatible with the tissue and cells of the tissue. The agitation
device is then partially inserted into the opening in the container
such that the impeller is submerged in the fluid. At least a
portion of the micromotor is exposed to the fluid and tissue during
operation. The micromotor is then connected to a voltage or current
source such that the motor shaft and impeller turn. The operator
applies sufficient voltage or current to mechanically agitate the
tissue such that single cells are disaggregated from the tissue
sample. Following sufficient disaggregation, the motor is
disconnected from the power source and the agitator device is
removed from the opening of the container. The fluid can then be
removed from the container, for example with a syringe or pipettor,
and the fluid, which contains the disaggregated single cells, can
be transferred to another container and then analyzed.
In some implementations, particulate material may be added to the
container. Such particulate material, e.g., ceramic or glass beads,
may aid in the mechanical disaggregation of the tissue sample.
The particulate material may take a variety of forms. While often
referred to herein as beads, the term bead is not meant to be
limiting with respect to size or shape. The beads may, for example,
comprise ceramic, glass, zirconia, zirconia/silica, zirconium
silicate, metal, plastic, nickel, tungsten, tungsten carbide,
yttrium stabilized zirconia, sand, and/or particles of any geometry
such as shard or of random shape.
In a first implementation, the disaggregation apparatus comprises a
container and an agitator comprising a micromotor attached to a
handle which may be comprised of plastic, metal, or any of a
variety of other solid materials. The micromotor is largely
cylindrical in shape having a shaft protruding from one of the ends
of the cylinder. The micromotor has two or more electrical wires
attached to supply power to the micromotor to induce the motor and
shaft to turn. The handle is mounted or attached to the end of the
micromotor opposite the end from which the shaft protrudes. The
handle is generally from one to three inches in length and provides
a means to handle the agitator conveniently. Attached to the shaft
of the micromotor is an impeller. The impeller generally has
blade-like protrusions from its generally cylindrical shape. The
blade-like protrusions may be of a wide variety of shapes and
geometries and generally aid in the transfer of mechanical energy
from the impeller to a fluid surrounding the impeller. The
disaggregation device further comprises a container having one
opening having a diameter slightly larger than the diameter of the
micromotor so that the micromotor may be slidingly engaged into the
opening. In some implementations, the container is a
microcentrifuge tube. The volume of the container is generally in
the range of 0.5 to 5 mL and more preferable 1 to 3 mL, for
example.
In operation, this disaggregation apparatus is used by inserting a
tissue sample and a fluid into the container and then inserting the
agitator micromotor into the opening in the container such that the
impeller is immersed in the fluid. A voltage or voltage waveform is
applied to the wire leads which induce a rotational motion of the
impeller which induces a turbulent rotational movement in the fluid
and tissue. The voltage or voltage waveform is applied for a period
of time sufficient to disaggregate the tissue into a suspension of
intact individual cells or small aggregates of cells without
causing substantial lysis of the cells of the tissue.
FIG. 1 shown one implementation of the disaggregation apparatus
described above. The handle 102 is attached to one end of the
micromotor 104. The shaft 105 protrudes from the micromotor and an
impeller 108 is attached to the shaft. Wire leads 110 and 112 are
attached to the motor to allow the application of a voltage or
voltage waveform to the micromotor. The leads are generally
attached to a connector (not shown) to allow simple attachment to
the voltage source. One lead, 110 is designated as negative and the
other lead, 112, is designated positive. When attached to a battery
having negative and positive terminals attached to the negative 110
wire lead positive wire leads, respectively, the motor shaft and
impeller will rotate in, for example a clockwise direction. If the
two wire leads are reversed, the rotation of the shaft and impeller
will be counter-clockwise. Application of a voltage waveform such
as a sine wave, triangle wave, square wave, or more complex
waveforms will result in changes in the rotation speed and
direction. The container 116 surrounds the micromotor and impeller.
In use the fluid and tissue fill the space in the container 116
surrounding the impeller and in contact with the micromotor
body.
The disaggregation apparatus or device of the type shown in FIG. 1
having an agitator comprising a handle and a micromotor and
impeller has dimensions which are small in comparison to prior art
devices for other applications which may comprise a motor and a
handle. For example, the dimensions of the device of FIG. 1 are
preferably only 25 to 75 mm in height and 5 to 15 mm in width. Such
small dimension is fortuitous in that the device may be used to
disaggregate small tissue samples using containers that have a
small (0.5 to 3 mL) volume.
The micromotors of the present disclosure are of the types that are
commonly used in cell phones to provide vibration alerts to the
user. The micromotors of the present disclosure are typically 4 mm
to 7 mm in diameter but can have diameters in the range of 3 mm to
10 mm, for example. Such micromotors work surprisingly well in this
application because they are small enough to fit into standard
microcentrifuge tubes. Further, the micromotors are able to operate
at a high speed (typically 20,000 to 50,000 rpm maximum speed) in
direct contact with fluid for a length of time sufficient to
perform disaggregation of tissue. The micromotors are also
advantageous in that they can be operated using batteries as the
voltage source since they draw only, for example, 20 to 100 mA of
current. Further, the micromotors are inexpensive enough that the
entire apparatus can be disposable after a single use or after
multiple uses.
In another preferred implementation, the disaggregation apparatus
comprises a container having a first opening for the introduction
of tissue and fluid and a second opening in which the micromotor is
sealingly engaged. The second opening is generally at the bottom of
the container such that gravity will pull the tissue and fluid into
contact with the micromotor, shaft, and impeller. This apparatus
generally has a base to support the opposite end of the micromotor
and thereby support the entire disaggregation apparatus. A lid for
the container is generally fitted into the first opening during the
disaggregation procedure so the fluid and tissue remain in the
container. Mounted on the shaft of the micromotor is an impeller.
Electrical leads or wires are connected to the micromotor to supply
a voltage or voltage waveform to induce the shaft and impeller to
rotate.
FIG. 2 shows another implementation of the apparatus or device. The
apparatus comprises a container 202 having a first opening 204 and
a second opening 206. A micromotor 208 is sealingly engaged in the
second opening 206 of the container 202. Protruding from the one
end of the micromotor is a shaft 210 upon which is mounted an
impeller 212. Electrically attached to the micromotor 208 are two
electrical leads or wires 214 and 216. The container 202 is mounted
in a solid base 218 which may be plastic or metal or another solid
material. In some implementations a permanent magnet is mounted in
the bottom of the base (not shown) to facilitate holding of the
apparatus on a ferromagnetic base plate (not shown). The first
opening 204 of the container 202 is optionally closed with a
removably and sealingly engaged cap 220 which seals the container
during use to contain a fluid and tissue during the disaggregation
process.
The disaggregation apparatus in its various implementations may be
combined with additional such devices in an array of devices to
form an apparatus for the disaggregation of multiple tissue samples
simultaneously of serially. For example, the agitator 100 of FIG. 1
may be combined with other such devices in a linear array device of
eight or twelve with a 9 mm spacing such that the linear array
disaggregation device can be used to disaggregate eight or twelve
tissue samples in, for example, one row or one column of a standard
96-well plate. In such an implementation, the plate replaces the
individual container of FIG. 1, serving as a 96-well container. The
96-well plate in one implementation is a deep-well plate which
better accommodates the depth of the multi-agitator when inserted
into the plate for disaggregation without spillage of the tissue or
fluid from the wells of the plate.
The disaggregation devices of the present disclosure, in many
implementations, use a micromotor which is sealed with a plastic
material or other material at the end opposite the shaft end of the
micromotor. It was discovered that sealing the end of the
micromotor facilitates operation of the micromotor in direct
contact with the fluid which contains the tissue. With the
non-shaft end of the motor sealed, the only opening to the interior
of the micromotor is around the shaft of the motor. Sealing of the
opposite end presumably helps to keep fluid from flowing into the
interior of the micromotor which could ultimately cause electrical
failure. In a test, it was shown that sealed micromotors can
function for several hours in contact with saline whereas
non-sealed micromotors only operated for a few minutes.
The devices of the intention are used to disaggregate tissue, in
one implementation using the device of FIG. 1 by placing the tissue
sample into the container. A buffer solution is also added to the
container which may be standard saline of a pH buffered saline of
any fluid compatible with the tissue and disaggregated cells. The
fluid is of sufficient volume to fully surround the tissue sample.
Then, the agitator is inserted into the container to a depth such
that the impeller is surrounded, at least partially, with the
fluid. A voltage or voltage waveform is then applied to the motor
through the wire leads. The motor is activated and turns its shaft
which in turn turns the impeller. The voltage or voltage waveform
is applied for a time sufficient to disaggregate the tissue into
intact single cells or multi-cell clusters suspended in the fluid.
The time required is typically from 30 seconds to 5 minutes. The
agitator is then removed from the container, leaving the
disaggregated tissue cell suspension in the container.
In some implementations, the voltage applied to the micromotor may
be important for efficient disaggregation of the tissue without
causing significant lysis of the cells. It has been shown that a DC
voltage can be employed where the voltage is usually less than the
recommended operating voltage for the particular micromotor being
used. For example, for a micromotor having a recommended operating
voltage of 1.5 V, it was found that a DC voltage of 0.25 to 1.5
volts would effectively disaggregate tissue. In some
implementations, voltages of 10% to 150% of the manufacturer
recommended operating voltage is applied and in at least some
implementations, a DC voltage of 25% to 75% of the manufacturer
recommended operating voltage is employed.
In other implementations of the present disclosure, voltage
waveforms are used to drive the micromotor. For example, if a
square wave is employed having a center voltage of zero and an
amplitude in the ranges described in the previous paragraph, the
micromotor will turn first in one direction and then in the other
direction. It is found that this waveform results in efficient
tissue disaggregation for some tissue types. In other
implementations, a sine wave or a triangle wave are used to drive
the micromotor. Any of the waveforms may have a zero or non-zero
center voltage and have an amplitude in the ranges described in the
previous paragraph.
The shape and dimensions of the impeller and container may also be
important to the efficient disaggregation of tissue samples. It was
found that an impeller in the shape of a cylinder having vanes
protruding from its surface may be advantageous. FIG. 3a shows a
cross section of an impeller 302 of one implementation of the
present disclosure furthest from the shaft of the micromotor. FIG.
3b shows a cross section of the same impeller 302 near the end
where the impeller is mounted on the shaft of the micromotor. A
cylindrical cavity 304 in the center of the impeller facilities
mounting of the impeller 302 on the cylindrical shaft of the
micromotor (not shown). Around the circumference of the impeller
are shown the vanes or blades 306 (only one numbered).
At least some of the implementations take advantage of the
understanding that the forces responsible for mechanical
disaggregation of biological samples such as tissue samples scale
with the oscillation frequency squared, and that by employing
relatively small sample sizes, the various implementations
described herein can achieve relatively higher frequencies as well
as lower frequencies than commercially available apparatus,
resulting in rapid and efficient tissue disaggregation.
In at least one implementation of the present disclosure, the
voltage source is integrated into the device. For example, for
implementations similar to that in FIG. 1, a battery, preferably in
the form of one or more button cells, may be included in the
handle. Such integration results in a device that is easier to use
by the end user. Further, the capacity of the voltage source may be
limited to discourage the device to be used for multiple
disaggregation procedures which can result in contamination by
carryover of components of one tissue sample to later disaggregated
tissue samples.
Example 1
Disaggregation of Chick Embryo Tissue
A small sample of chick embryo tissue was placed in phosphate
buffered saline (PBS). The tissue and PBS were transferred to the
disaggregation device of the type shown in FIG. 1. A DC voltage of
0.5 volts was applied to the micromotor for three minutes. The
agitator was removed from the micro-centrifuge tube and the
suspension was transferred to a clean tube and diluted with
Hibernate.RTM. media (Gibco). FIG. 4 shows photomicrographs of the
disaggregated suspension stained with Trypan Blue for visualization
(10.times. magnification). Also shown are 1:20 dilutions of the
cell suspension at both 10.times. and 40.times. magnification. The
cell suspension was stored in a refrigerator for five days and was
again observed under a microscope, as shown in the bottom right of
FIG. 4. Shown in FIG. 4 is a photomicrograph showing that the cells
remain healthy and intact.
Example 2
Disaggregation of Chick Heart Tissue
A small sample of chick heart tissue was placed in phosphate
buffered saline (PBS). The tissue and PBS were transferred to the
disaggregation device of the type shown in FIG. 1. A DC voltage of
0.5 volts was applied to the micromotor for three minutes. The
agitator was removed from the micro-centrifuge tube and the
suspension was transferred to a clean tube and diluted with
Hibernate.RTM. media (Gibco). FIG. 5, frame A, shows a
photomicrographs of the disaggregated suspension at 10.times.
magnification prior to dilution. FIG. 5, frame B, shows a
photomicrographs of the disaggregated suspension diluted 20:1 at
10.times. magnification. FIG. 5, frame C, shows a photomicrographs
of the disaggregated suspension diluted 20:1 at 40.times.
magnification. FIG. 5, frame D, shows a photomicrographs of the
disaggregated suspension diluted 20:1 at 40.times. magnification
following Trypan Blue staining which stains only dead cells.
The various embodiments described above can be combined to provide
further embodiments. U.S. Provisional Application 62/146,876, filed
Apr. 13, 2015 is incorporated herein by reference, in its entirety.
These and other changes can be made to the embodiments in light of
the above-detailed description. In general, in the following
claims, the terms used should not be construed to limit the claims
to the specific embodiments disclosed in the specification and the
claims, but should be construed to include all possible embodiments
along with the full scope of equivalents to which such claims are
entitled. Accordingly, the claims are not limited by the
disclosure.
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